Identification of the resistance mechanism to CDK4/6 inhibition and novel overcoming combination therapy with RNR inhibition for chemo-resistant bladder cancer

The identification of 42 down-regulated protein candidates in treated T24 cells and 46 in RT112 cells compared to the PBS control with 18 overlapping proteins (Supplementary Table S1); At the different time points, 2596, 3674 and 1994 genes were identified as differentially expressed genes (DEGs) (Supplementary Table S2, 3, 4); Identified activation of enormous genes that may confer acquired resistance to CDK4/6 inhibition after 7 days of CDK4/6 inhibition by a CRISPR-dCas9 activation functional genomics analysis, to further narrow down the functional candidates, we applied LFC＞1 and FDR＜0.001 and identified 365 potential candidates in this study (Supplementary Table S5); To further investigate the potential biological interaction network of RRM2 in clinical bladder cancer specimen, we inquired RRM2 in the TCGA cohorts for mRNA co-expression. Through this analysis, we identified the top 10 correlated genes: KIF18B, SKA3, FANCA, MYBL2, CENPA, FAM72B, IQGAP3, E2F1, FAM72D and CDT1 ranked by Spearman's Correlation (Supplementary Table S6); The informaton of prospectively collected 20 MIBC bladder cancer specimens from patients who received radical cystectomy in our hospital. Patients were divided into 3 subgroups according to therapy naïve, chemotherapy responsive and chemotherapy resistance (Supplementary Table S7)

与磷酸盐缓冲液（Phosphate Buffered Saline, PBS）对照组相比，处理后的T24细胞中鉴定出42个下调蛋白候选物，RT112细胞中鉴定出46个，其中18个为两者共有蛋白（补充表S1）；在不同时间点下，共鉴定出2596、3674和1994个差异表达基因（Differentially Expressed Genes, DEGs）（补充表S2、S3、S4）；本研究通过CRISPR-dCas9激活功能基因组学分析，发现经CDK4/6抑制剂（Cyclin-Dependent Kinase 4/6 Inhibitor, CDK4/6 Inhibitor）处理7天后，大量基因被激活，这些基因可能赋予细胞对CDK4/6抑制剂的获得性耐药；为进一步筛选功能候选基因，我们设置log2倍数变化（Log2 Fold Change, LFC）＞1且错误发现率（False Discovery Rate, FDR）＜0.001的筛选阈值，最终鉴定出365个潜在候选基因（补充表S5）；为进一步探究RRM2在临床膀胱癌标本中的潜在生物学相互作用网络，我们在癌症基因组图谱（The Cancer Genome Atlas, TCGA）队列中检索RRM2的mRNA共表达情况；通过该分析，我们基于斯皮尔曼相关性（Spearman's Correlation）排序得到前10个相关基因：KIF18B、SKA3、FANCA、MYBL2、CENPA、FAM72B、IQGAP3、E2F1、FAM72D及CDT1（补充表S6）；本研究前瞻性收集了我院接受根治性膀胱切除术的20例肌层浸润性膀胱癌（Muscle-Invasive Bladder Cancer, MIBC）患者标本；根据患者初始未治疗、化疗应答及化疗耐药情况，将其分为3个亚组（补充表S7）